Serotonin (5-HT)-producing neurons are recognized as key modulators of cocaine-seeking behavior. As cocaine-seeking behavior reflects the incentive motivational effect of stimuli paired with cocaine, 5-HT is thought to contribute to the formation of addiction-related memory that underlies relapse. Alteration of 5-HT commands is an emerging therapeutic direction for addiction treatment, with efforts focusing largely on receptor manipulations. Here we propose a novel, complementary approach that focuses presynaptically, on the 5-HT neurons themselves. Guided by recent research showing that cocaine seeking involves opposing activity of 5-HT at 5-HT2A and 5-HT2C receptors, which are key modulators of dopamine (DA) output, we will determine to what degree different subtypes of 5-HT neurons in mice differentially modulate the incentive motivational effects of stimuli paired with cocaine via the conditioned place preference procedure (Aim 1) and will explore their forebrain projection targets, especially as relates to postsynaptic 5-HT2A and 5-HT2C receptor expression (Aim 2). Our starting point is an emerging structure-function-connectivity map of the serotonergic neural system being assembled by the Dymecki lab, in which 5-HT neurons are classified by their expression of unique gene combinations and thus likely unique functionalities. They are further typed by their axonal target regions and by assessment of behavioral and physiological deficits following their selective silencing in vivo. Here we propose probing these 5-HT neuron subtypes for their role in addiction-related behavior, focusing first on 5-HT neuron subtypes that innervate brain regions implicated in enhancing or suppressing cocaine-seeking behavior. Three 5-HT neuron subtypes stand out: the r1-En1 5-HT neuron subtype, named by its origin in rhombomere (r) 1 and expression of the transcription factor Engrailed1; r2-Hoxa2 subtype, defined by its origin in r2 and expression of the transcription factor Hoxa2; and the Drd1a 5-HT neuron subtype, by expression of the type 1a DA receptor. Because the latter two show more restricted innervation profiles within the addiction-relevant mesolimbic system, perhaps suggestive of specialized roles in behavior modulation, these two will be explored first in this R21 application. Our approach of partnering molecular genetic techniques with a well-established behavioral assay in mice is both technically and conceptually innovative. The integration of these distinct disciplines is made possible only now through development of intersectional genetic tools that make molecularly distinct subtypes of 5-HT neurons apparent and accessible in mice for behavioral probing and genome-wide molecular profiling. Identifying the key 5-HT neuron subtypes involved and the nature of their effects on addiction-related behavior, along with having molecular genetic tools for their selective isolation and molecular probing is foundational for discovering new, possibly behaviorally-selective, therapeutic leads.